Compounds capable of inhibiting immunocyte-related allergic immune reactions

ABSTRACT

The present invention is based on novel discoveries relating to ebastine, carebastine, and epinastine hydrochloride. Specifically, the present invention relates to the use of ebastine and carebastine as an inhibitor of (A) T cell proliferation, (B) Th2 type cytokine production, (C) inflammatory cytokine production, and (D) T cell migration. In addition, it relates to the use of epinastine hydrochloride as an inhibitor of (A) T cell proliferation, (B) Th2 type cytokine production, and (C) Th1 type cytokine production.

TECHNICAL FIELD

The present invention relates to a novel application of ebastine,carebastine, and epinastine hydrochloride.

BACKGROUND ART

Allergic diseases are caused by overreaction of the immune system. Basicallergic reactions are generally classified into four types depending ontheir mechanism. Type I (immediate) allergy, also called anaphylaxis,mainly involve IgE antibodies. Damage to one's own tissue (self-tissuedamages)(type II allergy) due to abnormal antibodies (autoantibodies)that can react with one's own cells or tissues involve IgG, IgM, IgA,etc. Immune complex disease is caused by type III allergy that involveantigen-antibody complexes comprising antibodies such as IgG, IgM, andIgA, antigens, and complements. Type IV (delayed type) allergy does notinvolve antibodies but is caused by reactions between T cells and Tcell-derived inflammatory substances. In addition, among type IIallergy, those that involve receptor-stimulated immunoglobulins are alsocalled type V allergy.

Type I (immediate) allergy represented by urticaria or allergic rhinitisrequire the production of antigen-specific IgE in advance. Foreignantigens entering the body are first incorporated into antigenpresenting cells such as macrophages and dendritic cells. These antigensare degraded into peptides of 11 to 13 amino acids in endosomes,presented on the cell membrane by binding to MHC class II molecules, andare then recognized by T cells. When a T cell recognizes a complex ofMHC and a peptide via the T cell antigen receptor (TCR), a signal istransmitted into the cell activating them to produce various cytokines.B cells differentiate and maturate into plasma cells due to the antigenstimulation and produce antibodies (immunoglobulins) that are specificfor particular antigen(s). In the peripheral tissue, depending on theenvironment, naïve T cells differentiate into Th1 type or Th2 typesubsets. Cytokines IL-4 and IL-5 that are produced by the Th2 typesubset act on B cells to direct the production of IgE and IgG1.

The binding of a foreign antigen-specific IgE antibody produced by a Bcell to a high-affinity IgE receptor (FcεRI) of a mast cell or basophilalone induces transmission of activation signal into the nucleus,enhances the expression of FcεRI, and suppresses apoptosis to extend thelifetime of the mast cell or basophil. Moreover, when a foreign antigenbinds to FcεRI, degranulation arises and histamine and leukotriene arereleased that act on the surrounding tissues (exalting vascularpermeability, contracting smooth muscle, etc.), thereby causing varioussymptoms of allergy. However, recent studies have indicated chronicinflammation as the main condition of the diseases in many type Iallergic diseases such as bronchial asthma.

Chronic allergic inflammations that follow immediate reaction are causedby the migration and infiltration into the inflammation area ofinflammation-related cell group, such as eosinophils, neutrophils, andlymphocytes that are activated by cytokines that are mainly released bylocal mast cells. The role of lymphocytes is thought to be indispensablein prolonging the inflammation of immediate allergic reactions.

T-cell activation by antigen induces phosphorylation of intracellularprotein and elevation of intracellular calcium concentration at an earlystage and is complete within several minutes from the antigenstimulation. After 24 to 48 hours, various activation antigen moleculesand IL-2 receptor are expressed on the T cell surface, and cytokines arecopiously produced and released to trigger cell proliferation reactions.

Hypersensitive allergic immune response is induced and continued by manytypes of inflammatory cells through the production of soluble factors,such as cytokines and intercellular interactions. In particular, helperT cells (hereinafter abbreviated as “Th cells”) are thought to be theeffector cells and play an important role in controlling hypersensitivereactions. Two subsets of helper T cells (CD4⁺ T cells) have beenidentified based on their cytokine production profile (Kapsenberg M,Wierenga E, Bos J, Jansen H. “Functional subsets of allergen-reactivehuman CD4⁺ T cells.” Immunol Today. 12:392-395, 1991; Romagnani S.“Lymphokine production by human T cell in disease states.” Ann RevImmunol. 12:227-257, 1994).

One is the T helper cell type 1 (Th1) which releases IL-2, IFN-γ, and soon (Kapsenberg M. et al., 1991, supra; Romagnani S., 1994, supra). Theother is the T helper cell type 2 (Th2) which releases IL-4, IL-5, andso on (Kapsenberg M. et al., 1991, supra; Romagnani S., 1994, supra).Inflammatory cytokines (IL-6, TNF-α, etc.) are also produced bymacrophages and the like in addition to T cells.

IL-4 acts on B cells to promote class switching, thereby playing animportant role in IgE production. On the other hand, IL-2 and IFN-γsuppress IgE production enhancing the action of IL-4 in a differentmanner respectively. In addition to cytokines, stimulation of the B cellsurface molecule CD40 is important in inducing the production of IgE.During this process, B cell proliferation is induced by IL-6, and IL-5acts in an auxiliary manner. Furthermore, IL-5 specifically induces thedifferentiation and proliferation of eosinophils, and promotes mediatorsecretion from eosinophils and basophils. In several allergic diseases,it has been reported that Th2 cells preferentially accumulate atinflammatory sites and induce hypersensitive reactions (Del Prete G F,De Carli M, D'Elios M M et al. “Allergen exposure induces the activationof allergen-specific Th2 cells in the airway mucosa of patients withallergic respiratory disorders.” Eur J Immunol. 23:1445-1449, 1993;Robinson D, Hamid Q. Bentley A, Ying S, Kay A B, Durham S R. “Activationof CD4⁺ T cells, increased Th-2 type cytokine mRNA expression, andeosinophil recruitment in bronchoalveolar lavage after allergeninhalation challenge in patients with atopic asthma.” J Allergy ClinImmunol. 92:313-324, 1993; Maggi E, Biswas P, Del Prete G P Parronchi P,Nacchia D, Simonelli C, Emmi L, Decarli M, Tiri A, Ricci M, et al.“Accumulation of Th2-like helper T cells in the conjunctiva of patientswith vernal conjunctivitis.” J Immunol. 146: 1169-1174, 1991).Therefore, it is considered possible to treat such allergic diseases andhypersensitivity by specifically inhibiting the function of Th2 cells.

Macrophages not only present antigens in the initial phase, but also getself-activated to actively contribute to the proliferation,differentiation, and activation of neighboring cells via production ofinflammatory cytokines (IL-6, TNF-α, and so on). These various cytokinesproduced by macrophages also play important roles in allergic reactions.

The migration and infiltration of inflammatory cells at inflammatorysites have important roles in allergic reactions. The lymphocyteextravasation consists of three steps, i.e., the cell adhesion cascade:(1) rolling, (2) strong adhesion, and (3) transmigration. Each of thesteps is mediated by the binding of adhesive molecules and ligandsexpressed on the cell membranes of leukocytes and vascular endothelialcells, respectively.

The adhesive molecules are structurally categorized into a large numberof families: the integrin family, the immunoglobulin superfamily, theselectin family, the cadherin family, the link protein family, thesialomucin family, etc.

Surface molecules present on activated T cells, such as CD11a, CD29,CD44, CD26, and CD47, have a close relationship with cell adhesion.CD11a has been revealed to be the α chain of LFA-1. LFA-1 is expressedonly in lymphocytes within lymphatic tissue, and is associated withintercellular adhesion via the binding with its ligand ICAM-1. Among theβ1 integrin family members, VLA-4 (CD49d/CD29) is particularly involvedin the adhesion of T cells, and one of its ligands is VCAM-1 that isexpressed on vascular endothelial cells. CD44 is a hyaluronic acidreceptor that is associated with the adhesion of lymphocytes toendothelial cells and interstitial cells at inflammatory sites. CD26 isexpressed on memory T cells and is associated with the migration of Tcells (Hafler D A, Fox D A, Manning M E, Schlossman S F, Reinherz E L,Weiner H L. “In vivo activated T lymphocytes in the peripheral blood andcerebrospinal fluid of patients with multiple sclerosis.” N Engl J Med.312:1405, 1985; Nakao H, Eguchi K, Kawakami A, Migita K, Otsubo T, UekiY, et al. “Increment of Ta1 positive cells in peripheral blood frompatients with rheumatoid arthritis.” J Rheumatol. 16:904, 1989). CD47 iscalled integrin-associated protein and is mainly involved in cellmigration (Masuyama J, Berman J S, Cruikshank W W, Morimoto C, Center DM. “Evidence for recent as well as long term activation of T cellsmigrating through endothelial cell monolayers in vitro.” J Immunol.148:1367, 1992; Ohashi Y, Iwata S, Kamiguchi K, Morimoto C. “Tyrosinephosphorylation of Crk-associated substrate lymphocyte-type is acritical element in TCR- and beta 1 integrin-induced T lymphocytemigration.” J Immunol. 163:3727, 1999; Liu Y, Merlin D, Burst S L,Pochet M, Madara J L, Parkos C A. “The role of CD47 in neutrophiltransmigration. Increased rate of migration correlates with increasedcell surface expression of CD47.” J Biol. Chem. 276:40156, 2001).

Moreover, β1 integrin is not only involved in adhesion and migration ofcells but also has various biological functions including activation andproliferation, and cytokine production of T cells. Both β1 integrins andextracellular matrix accumulate upon binding of the two, followed byaccumulation of intracellular tyrosine kinase (FAK, Src), adapterproteins (p130Cas, paxillin), and actin-binding proteins (α-actinin,vinculin, talin) (Schaller M D, Otey C A, Hildebrand J D, Parsons J T.“Focal adhesion kinase and paxillin bind to peptides mimicking betaintegrin cytoplasmic domains.” J Cell Biol. 130:1181, 1995; Lewis J M,Schwartz M A. “Mapping in vivo associations of cytoplasmic proteins withintegrin beta 1 cytoplasmic domain mutants.” Mol Biol Cell. 6(2):151,1995). These accumulated protein aggregates bind to actin fibers to formfocal adhesions that repeatedly activate target molecules through enzymereactions such as tyrosine phosphorylation, in order to controlbiological functions via generation of various signals.

The present inventors have reported strong tyrosine phosphorylation of a105-kDa protein (pp105) in T cells through the stimulation mediated byβ1 integrin (Nojima Y, Rothstein D M, Sugita K, Schlossman S F, MorimotoC. “Ligation of VLA-4 on T cells stimulates tyrosine phosphorylation ofa 105-kD protein.” J Exp Med. 175:1045, 1992). Through cDNA cloning,pp105 was revealed to be a homologue of Crk-associated substrate. Thus,this protein was named Crk-associated substrate lymphocyte type (Cas-L)(Minegishi M, Tachibana K, Sato T, Iwata S, Nojima Y, Morimoto C.“Structure and function of Cas-L, a 105-kD Crk-associatedsubstrate-related protein that is involved in beta 1 integrin-mediatedsignaling in lymphocytes.” J Exp Med. 1:1365, 1996; Law S F, Estojak J,Wang B, Mysliwiec T, Kruh G, Golemis E A. “Human enhancer offilamentation 1, a novel p130-like docking protein, associates withfocal adhesion kinase and induces pseudohyphal growth in Saccharomycescerevisiae.” Mol Cell Biol. 16:3327, 1996). In addition, Cas-L was foundto be directly tyrosine phosphorylated by FAK and Src family tyrosinekinases (Tachibana K, Urano T, Fujita H, Ohashi Y, Kamiguchi K, Iwata S,et al. “Tyrosine phosphorylation of Crk-associated substrates by focaladhesion kinase. A putative mechanism for the integrin-mediated tyrosinephosphorylation of Crk-associated substrates.” J Biol. Chem. 272:29083,1997). Cas-L is expressed in lymphatic cells such as peripheral blood Tcells and B cells, and thymus cells. Cas-L is tyrosine phosphorylated byv-Src, BCR-ABL, and v-Crk molecules, and binds to adapter proteins suchas Crk and Nck, tyrosine kinases such as Src, and phosphatases such asSH-PTP2 via their SH2 domains. Tyrosine phosphorylation of Cas-L isessential for T cell activation mediated by β1 integrin and plays animportant role in T cell proliferation, cytokine production, cellmigration, and cell adhesion (Ohashi Y, Iwata S, Kamiguchi K, MorimotoC. “Tyrosine phosphorylation of Crk-associated substrate lymphocyte-typeis a critical element in TCR- and beta 1 integrin-induced T lymphocytemigration.” J Immunol. 163:3727, 1999; Kamiguchi K, Tachibana K, IwataS, Ohashi Y, Morimoto C. “Cas-L is required for beta 1 integrin-mediatedcostimulation in human T cells.” J Immunol. 163:563, 1999). Accordingly,studies of tyrosine phosphorylation of β1 integrin-related moleculesrepresented by Cas-L are thought to be important in the evaluation of Tcell functions such as cell adhesion and cell migration.

Histamine is an in vivo amine intracellularly synthesized fromL-histidine by a decarboxylase, a compound isolated by Dale in 1910 fromergot. Histamine is mainly stored within granules of mast cells andbasophils. It is released by various physical and chemical stimuli andthe cross-link formation of IgE-FcεRI complex by antigens. Variousphysiological activities of histamine including immune-related reactionsin target tissue such as acceleration of vascular permeability,contraction of smooth muscles, vasodilation, and elevation of mucussecretion, as well as acceleration of gastric acid secretion fromgastric parietal cells have been well studied. Recently, histamine hasbeen found to act as an important neurotransmitter in the centralnervous system. These physiological activities are expressed via ahistamine receptor.

Four subtypes of histamine receptors (H1, H2, H3, and H4) have currentlybeen identified. The H1 receptor is mainly expressed on tissues such ascapillaries, vascular smooth muscles, vascular endothelia, bronchialsmooth muscles, intestinal tract smooth muscles and so on. It causestypical immediate allergic symptoms via the binding of histaminesreleased from mast cells. The H2 receptor that is mainly expressed ongastric parietal cells and airway goblet cells, secrete gastric acid andairway mucus due to histamine stimulation. Within the central nervoussystem, there is a nervous system called the histamine nervous systemwhere the histamine receptor subtypes H1, H2, and H3 are expressed. Inaddition to the nerve cells, the H1 and H2 receptors are also expressedon glial cells. The H1 receptor of the histamine nervous system mediatevarious central functions including awakening, appetite, drinking, bodytemperature regulation, regulation of the sense of equilibrium,regulation of neuroendocrine, and suppression of convulsions. The mainside effect of histamine H1 receptor antagonists (i.e., H1 blockers) isdrowsiness, because the H1 receptor in the brain controls awakening. TheH3 receptor is mainly expressed in the central nervous system and hasbeen identified as an autoreceptor that controls histamine release fromhistamine nerve endings. Moreover, it is also expressed on the nerveendings of nerve systems other than the histamine nerve system, and issuggested to be involved in the suppression of transmitter release. Theexpression of the H4 receptor has been confirmed in peripheral tissuesand their functions are currently being analyzed.

Histamine has recently been reported to participate in the regulation ofallergy and inflammation (Jutel M, Watanabe T, Klunker S, Akdis M,Thomet O A, Malolepszy J, et al. “Histamine regulates T-cell andantibody responses by differential expression of H1 and H2 receptors.”Nature. 413:420-5, 2001; Jutel M, Klunker S, Akdis M, Malolepszy J,Thomet O A, Zak-Nejmark T, et al. “Histamine upregulates Th1 anddownregulates Th2 responses due to different patterns of surfacehistamine 1 and 2 receptor expression.” Int Arch Allergy Immunol.124(1-3):190-2, 2001; Lagier B, Lebel B, Bousquet J, Pene J. “Differentmodulation by histamine of IL-4 and interferon-gamma (IFN-gamma) releaseaccording to the phenotype of human Th0, Th1 and Th2 clones.” Clin ExpImmunol. 108(3):545-51, 1997; Horvath B V, Szalai C, Mandi Y, Laszlo V,Radvany Z, Darvas Z, et al. “Histamine and histamine-receptorantagonists modify gene expression and biosynthesis of interferon-gammain peripheral human blood mononuclear cells and in CD19-depleted cellsubsets.” Immunol Lett. 70(2):95-9, 1999). The H2 receptor is presentmainly on human T cells (Sachs B, Hertl M, Merk H F. “Histaminereceptors on lymphocytes, distribution and functional significance.”Skin Pharmacol Appl Skin Physiol. 13(6):313-23, 2000). However,histamine H1 and H2 receptors are present on mouse T cells and arereported to be involved in signal transduction for promoting immuneactivation and suppressing immune activation, respectively. A hypothesishas been proposed that histamine enhances Th1 cytokine production from Tcells via the H1 receptor and at the same time suppresses both Th1 andTh2 cytokine production from T cells via the H2 receptor (Jutel M, etal., Nature, 2001, supra).

Histamine H1 receptor antagonists developed as agents to treat immediateallergy strongly bind to the H1 receptor of target cells, preventingbinding of histamine to the receptor and suppressing allergic symptoms.In addition, they also have various pharmacological effects such aslocal anesthetic effects, central nervous system sedative effects,anti-arrhythmic effects, atropine-like effects, anti-serotonin effects,and anti-kinin effects. Aiming to reduce side effects and increase drugcompliance, hitherto, a large number of first- and second-generation H1blockers (antihistamines) have been developed. The second-generation H1blockers have less sedative effects such as drowsiness andanti-cholinergic effects. Therefore, they are presently widely used astherapeutic agents for type I allergic diseases such as urticaria andallergic rhinitis (Storms W W. “Clinical studies of the efficacy andtolerability of ebastine 10 or 20 mg once daily in the treatment ofseasonal allergic rhinitis in the US.” Drugs. 52 Suppl 1:20, 1996; KalisB. “Double-blind multicentre comparative study of ebastine, terfenadineand placebo in the treatment of chronic idiopathic urticaria in adults.”Drugs. 52 Suppl 1:30, 1996).

H1 blockers have an ammonium group common to their chemical structureand are recognized via this ammonium group by H1 receptors. On the otherhand, other pharmacological effects are determined based on thestructure other than this ammonium group. H1 blockers also bind to theH2 receptor, albeit to a lesser extent than their binding to the H1receptor (Leurs R, Church M K, Taglialatela M. “H1-antihistamines:inverse agonism, anti-inflammatory actions and cardiac effects.” ClinExp Allergy. 32(4):489-98, 2002). In addition, a large number ofhistamine antagonists have been reported to show inverse agonism (PillotC, Ortiz J, Heron A, Ridray S, Schwartz J C, Arrang J M. “Ciproxifan, ahistamine H3-receptor antagonist/inverse agonist, potentiatesneurochemical and behavioral effects of haloperidol in the rat.” Journalof Neuroscience. 22(16):7272-80, 2002; Nonaka H, Otaki S, Ohshima E,Kono M, Kase H, Ohta K, et al. “Unique binding pocket for KW-4679 in thehistamine H1 receptor.” Eur J Pharmacol. 345:111-7, 1998). Depending onthe structure of the H1 blockers, differences have been recognized intheir affinity to the H1 blocker receptors and in their clinicaleffectiveness.

Terfenadine is an agent that can suppress the aforementioned Th2 typecytokine production. This agent is an H1 blocker that does not induceanticholinergic effects or drowsiness. However, it has been reported toinfrequently cause side effects including QT interval prolongation andarrhythmia. These side effects are considered to be the result of itsantagonism to histamine receptors (Roberts D J. “A preclinical overviewof ebastine. Studies on the pharmacological properties of a novelhistamine H1 receptor antagonist.” Drugs 52 (Suppl):8-14, 1996).However, recent researches suggest that terfenadine may have differentproperties that are unrelated to histamine blocking at the receptorlevel (Massey W A, Lichtenstein L M. “The effect of antihistaminesbeyond H1 antagonism in allergic inflammation.” J Allergy Clin Immunol.86:1019-1024, 1990; Crampette L, Mainprice B, Bloom M, Bousquet J,Campbell A M. “Inhibition of mediator and cytokine release fromdispersed nasal polyp cells by terfenadine.” Allergy. 51:346-349, 1996).

H1 blockers that similarly induce no anticholinergic effects ordrowsiness include ebastine(4-diphenylmethoxy-1[3-(4-terbutyl-benzoyl)-propyl]piperidine; molecularweight 471.68). Ebastine is structurally similar to terfenadine but hasalmost no side effect such as QT interval prolongation and arrhythmia.Therefore, it is widely used today as a therapeutic agent for seasonalallergic rhinitis and the like (Storms W W. “Clinical studies of theefficacy and tolerability of ebastine 10 or 20 mg once daily in thetreatment of seasonal allergic rhinitis in the US.” Drugs 52 (Suppl.1):20-25, 1966; Kalis B. “Double-blind multicentre comparative study ofebastine, terfenadine and placebo in the treatment of chronic idiopathicurticaria in adults.” Drugs 52 (Suppl. 1):30-34, 1996). Althoughebastine is effective in the treatment of allergic diseases, its Th2type cytokine production-suppressing activity and such has not beenanalyzed. Hence, it may include other mechanisms.

In the initial stages of a hypersensitive response, specific allergensare recognized by T cell receptors (TcR), whereby T cells get activatedand release various cytokines that induce allergy. However, this processalone cannot induce the activation of T cells. Various findings suggestthe existence of a second factor called costimulation signal inducedthrough an additional T cell surface molecule different from TcR (RobeyE, Allison J P. “T-cell activation: Integration of signals from theantigen receptor and costimulatory molecule.” Immunol Today. 16:306-309,1995).

The costimulation signal is characterized by T cell proliferation,cytokine production and so on. In fact, this signal is essential for acomplete activation of T cells via TcR stimulation. Therefore, theinduction of a costimulation signal also plays an important role inhypersensitive immune reactions. The costimulation signal may beproduced by several types of accessory molecules such as CD28/CTLA-4(Green J M, Noel P J, Sperling A I, Walunas T L, Leishow D J, Stack R,Gray G S, Bluestone J A, Thompson C B. “Absence of B7-dependentresponses in CD28-deficient mice.” Immunity. 1:501-508, 1994; Gimmi C D,Freeman G J, Sugita K, Freedman A S, Morimoto C, Nadler L N. “B7provides a costimulatory signal which induces T cells to proliferate andsecrete IL-2.” Proc Natl Acad Sci USA. 88:6575-6579, 1991; Freeman G J,Borriello F, Hodes R J, Reiser H, Hathcock K S, Laszlo G, McKnight A J,Kim J, Du L, Lombard D B, et al. “Uncovering of functional alternativeCTLA-4 counter-receptor in B7-deficient mice.” Science. 262:907-909,1993; Linsley P S, Ledbetter J A. “The role of the CD28 receptor duringT cell response to antigen.” Annu Rev Immunol. 11:191-212, 1993).

Moreover, the present inventors have recently identified a novelcostimulation molecule, CD26, that is expressed on CD4⁺ memory T cells(Morimoto C, Torimoto Y, Levinson G, Rudd C E, Schrieber M, Dang N H,Letvin N L, Schlossman S F. “1F7, a novel cell surface molecule involvedin helper function of CD4 cells.” J Immunol. 143:3430-3439, 1989; Dang NH, Torimoto Y, Deusch K, Schlossman S F, Morimoto C. “Comitogenic effectof solid-phase immobilized anti-1F7 on human CD4 T cell activation viaCD3 and CD2 pathways.” J Immunol. 144:4092-4100, 1990; Tanaka T, KameokaT, Yarson A, Schlossman S F, Morimoto C. “The costimulatory activity ofthe CD26 antigen requires dipeptidyl peptidase IV enzyme activity.” ProcNatl Acad Sci USA. 90:4586-4590, 1993). In addition, this CD26 moleculeis also said to be involved in the migration of effector T cells toinflammatory sites; such migration is seen in diseases caused byimmunity (Hafler D A, Fox D A, Manning M E, Schlossman S F, Reinherz EL, Weiner H L. “In vivo activated T lymphocytes in the peripheral bloodand cerebrospinal fluid of patients with multiple sclerosis.” N Engl JMed. 312:1405-1411, 1985; Nakao H, Eguchi K, Kawakami A, et al.“Increment of Ta1 positive cells in peripheral blood from patients withrheumatoid arthritis.” J Rheumatol. 16:904-910, 1989).

DISCLOSURE OF THE INVENTION

The first objective of the present invention is to elucidate a novelaction mechanism of ebastine(4-diphenylmethoxy-1[3-(4-terbutyl-benzoyl)-propyl]piperidine),carebastine (4-[4-[4-(di-phenylmethoxy)-1-piperidinyl]-1-oxobutyl]-α,α-di-methylbenzeneacetic acid), and epinastine hydrochloride(3-amino-9,13b-dihydro-1H-dibenz(c,f)imidazol(1,5-a)azepinehydro-chloride; molecular weight 285.78) (Roeder T, Degen J, Gewecke M.“Epinastine, a highly specific antagonist of insect neuronal octopaminereceptors.” Eur J Pharmacol, 349(2-3):171-177, 1998). Another objectiveof the present invention is to provide compounds having fewer sideeffects and that are able to suppress allergic immune response due tocostimulation signals such as cytokine production and T cell migrationas described above. Yet another objective of the present invention is toprovide compositions comprising these compounds.

In order to achieve the aforementioned objectives, the present inventorsattempted to analyze the effects of ebastine, carebastine, andepinastine hydrochloride on T cell proliferation and cytokine productionvia different costimulation signal pathways in order to analyze theallergic immune response inhibition mechanism of ebastine, carebastine,and epinastine hydrochloride. Furthermore, the effects of ebastine,carebastine, and epinastine hydrochloride on T cell migration andinflammatory cytokine production due to T cells and macrophages wereanalyzed. These analyses indicated that under costimulation conditions,ebastine and carebastine inhibit T cell proliferation, Th2 type cytokineproduction, inflammatory cytokine production, as well as T cellmigration, all in a concentration-dependent manner. In addition,epinastine hydrochloride was indicated to inhibit, in aconcentration-dependent manner, T cell proliferation, Th2 type cytokineproduction, and Th1 type cytokine production. The present invention isbased on these novel findings relating to ebastine, carebastine, andepinastine hydrochloride. Specifically, the present invention provides:

-   -   [1] a compound having the formula (1):        (wherein, R is CH₃ or COOH),        that is able to suppress an allergic immune response involving        an immunocyte, wherein said allergic immune response is selected        from the group consisting of:    -   (A) proliferation of T cell;    -   (B) production of Th2 type cytokine;    -   (C) production of inflammatory cytokine; and    -   (D) T cell migration;    -   [2] a compound having the formula (2) or salts thereof:        that is able to suppress an allergic immune response involving        an immunocyte, wherein said allergic immune response is selected        from the group consisting of:    -   (A) proliferation of T cell;    -   (B) production of Th2 type cytokine; and    -   (C) production of Th1 cytokine;    -   [3] the compound according to [1], wherein the inflammatory        cytokine is produced from a T cell or macrophage;    -   [4] a composition comprising the compound according to [1] or        [3];    -   [5] a composition comprising the compound or salts thereof        according to [2];    -   [6] an agent for suppressing an allergic immune response        involving an immunocyte that comprises, as an active ingredient,        the compound having the formula (1):        wherein said allergic immune response is selected from the group        consisting of:    -   (A) proliferation of T cell;    -   (B) production of Th2 type cytokine;    -   (C) production of inflammatory cytokine; and    -   (D) T cell migration; and    -   [7] an agent for suppressing an allergic immune response        involving an immunocyte that comprises, as an active ingredient,        the compound having the formula (2) or salts thereof:        wherein said allergic immune response is selected from the group        consisting of:    -   (A) proliferation of T cell;    -   (B) production of Th2 type cytokine; and    -   (C) production of Th1 type cytokine.

The present invention is based on the discovery of novel functions ofebastine (the compound of formula (1) above, wherein R is CH₃) and itsoxidized metabolite, carebastine (the compound of formula (1) above,wherein R is COOH), and relates to the use of ebastine and carebastineas agents that suppress allergic immune response involving immunocytesother than as H1 blocker. In addition, the present invention relates tothe use of ebastine and carebastine in methods for treating diseasesassociated with allergic immune response involving immunocytes thatcomprise the administration of ebastine or carebastine, or in themanufacture of agents that suppress allergic immune response involvingimmunocytes.

Examples of the novel functions of ebastine and carebastine include:suppression of T cell proliferation, suppression of Th2 type cytokineproduction, suppression of inflammatory cytokine production from T cellsor macrophages, and suppression of T cell migration. That is, thepresent invention relates to the use of ebastine and carebastine asagents suppressing the aforementioned various allergic immune responsesinduced by immunocytes comprising T cells and macrophages that play acentral role in allergic immune response. Herein below, each of thesevarious functions is described in detail.

The suppression of T cell proliferation can be mentioned as the firstexample of the novel function of ebastine and carebastine. T cellproliferation is a phenomenon wherein the number of T cells increasethrough, for example, activation of T cells in the resting stage due toexogenous or autoantigen stimuli that are able to induce allergy. Such Tcell proliferation can be measured in cell cultures, for example, by theintake of ³H-thymidine and so on. The in vitro ebastine concentrationrequired to suppress such T cell proliferation is 1 μM to 20 μM,preferably 10 μM to 20 μM. In addition, the in vitro carebastineconcentration required to suppress the T cell proliferation is 1 μM to200 μM, preferably 20 μM to 200 μM.

As the second example of the novel function of ebastine and carebastine,the ability to selectively suppress the production of Th2 type cytokinescan be mentioned. Herein, “selectively” means that the production of Th2type cytokines detected at inflammatory sites in allergic diseases aloneare specifically suppressed rather than suppressing the production ofall cytokines from T helper cells, without affecting the production ofcytokines such as IL-2 and TNF-γ from Th1 type cells. Th2 type cytokinesare those produced from T helper cell type 2, which is detected atinflammatory sites of a wide range of immunoallergic diseases includingbronchial asthma, atopic dermatitis, chronic graft-versus-host disease(GVHD), systemic lupus erythematosus, and myasthenia gravis. Examples ofthese cytokines include IL-4, IL-5, IL-6, IL-10, and IL-13. Theidentification and quantification of these Th2 type cytokines can beperformed by ELISA wherein antibodies against the cytokines areimmobilized. The in vitro ebastine concentration required to suppressthe production of Th2 type cytokines is 1 μM to 20 μM, preferably 5 μMto 20 μM, and even more preferably 10 μM. In addition, the in vitrocarebastine concentration required to suppress the production of Th2type cytokines is 1 μM to 200 μM, preferably 10 μM to 200 μM, and evenmore preferably 100 μM to 200 μM.

The third novel function of ebastine and carebastine is exemplified bythe suppression of inflammatory cytokine production. Examples of theseinflammatory cytokines typically include IL-1 and IL-6; however herein,they are not limited thereto and include TNF-α and so on. Furthermore,the inflammatory cytokines whose production is suppressed by ebastineand carebastine comprise not only those produced from macrophages butalso those produced from T cells. The ebastine concentration required tosuppress the production of inflammatory cytokines by T cells in an invitro system is 1 μM to 20 μM, preferably 5 μM to 20 μM, and even morepreferably 10 μM. Moreover, the ebastine concentration required tosuppress the production of inflammatory cytokines by macrophages in anin vitro system is also 1 μM to 20 μM, preferably 5 μM to 20 μM, andeven more preferably 10 μM. In addition, the carebastine concentrationrequired to suppress the production of inflammatory cytokines by T cellsin an in vitro system is 1 μM to 200 μM, preferably 10 μM to 200 μM, andeven more preferably 100 μM to 200 μM. Moreover, the carebastineconcentration required to suppress the production of inflammatorycytokines by macrophages in an in vitro system is 1 μM to 200 μM,preferably 10 μM to 200 μM, and even more preferably 100 μM to 200 μM.The measurement of inflammatory cytokines such as IL-6 and TNF-α can beperformed, for example, using an ELISA kit wherein antibodies to thecorresponding cytokines are immobilized.

T cell migration can be mentioned as the fourth novel function ofebastine and carebastine. In the initial stage of the onset ofinflammation in an allergic disease, T cells permeate through vascularendothelial cells and such, and accumulate at inflammatory sites. Theaforementioned Th2 type cytokines, inflammatory cytokines, and such arethen released from the accumulated T cells to induce inflammation.Ebastine and carebastine inhibit transendothelial T cell migration inthe initial stage of allergic immune response. The ebastineconcentration required to inhibit T cell migration in an in vitro systemis 1 μM to 10 μM, preferably 5 μM. In addition, the carebastineconcentration required to inhibit T cell migration in an in vitro systemis 1 μM to 200 μM, preferably 100 μM to 200 μM. The migration of T cellscan be specifically measured by the method described in Example 5. Putsimply, T cells are placed on one side of a layer of vascularendothelial cells and then the number of T cells that have moved to theother side is counted after a predetermined time.

The concentrations of ebastine and carebastine required to suppress thevarious allergic immune responses described above are those that exhibitactivity in vitro. However, the concentrations used in vivo (in animalsand the like) and treating human diseases can be readily determined bythose skilled in the art using the aforementioned in vitroconcentrations as a standard or reference, and using the analyticalmethods mentioned above or presented in the Examples.

As described above, ebastine and carebastine can be used to suppressallergic immune response, such as T cell proliferation, Th2 typecytokine production, inflammatory cytokine production by T cells andmacrophages, and T cell migration, as well as inflammatory reactionsarising from allergic immune response. Therefore, ebastine andcarebastine can be used as agents to treat diseases involving theseallergic immune responses.

Examples of such diseases include allergic rhinitis, bronchial asthma,atopic dermatitis, systemic lupus erythematosus, myasthenia gravis,chronic GVHD, and Th2 type autoimmune diseases (e.g., lupus nephritis).

In diseases like bronchial asthma, T cells have been found to migrateand accumulate at inflammatory sites to produce Th2 type cytokines atthese sites. As described above, ebastine and carebastine suppress Tcell migration and further suppress Th2 type cytokine production.Therefore, ebastine and carebastine are applicable as pharmaceuticalsfor the treatment of asthma and other diseases that involve such Th2type cytokines.

Furthermore, in inflammatory diseases such as atopic diseases,inflammatory cytokines are detected at inflammatory sites. Ebastine andcarebastine can suppress the production of inflammatory cytokines fromboth the macrophages and the T cells. Therefore, ebastine andcarebastine can be effectively used as agents for the treatment ofallergic inflammatory diseases that involve inflammatory cytokines, suchas atopic diseases. Moreover, TNF-α and IL-6 antibody treatment is saidto be effective in treating chronic rheumatoid arthritis and theinflammatory bowel disease, Crohn's disease, and the like. Hence,ebastine and carebastine may be used as agents to treat these diseaseseither alone or in combination with antibody treatment.

Furthermore, the present invention is based on the discovery of novelfunctions of the compound of formula (2) or salts thereof, and relatesto the use of this compound as an agent other than an H1 blocker thatsuppresses allergic immune response involving immunocytes. In addition,the present invention relates to the use of the compound of formula (2)or salts thereof in methods of treating diseases associated withallergic immune response involving immunocytes, wherein the methodscomprise administering the compound of formula (2) or salts thereof, orin the manufacture of agents that suppress allergic immune responseinvolving immunocytes. The salts according to the present inventionpreferably include hydrochloride (epinastine hydrochloride); however, itis in no way limited thereto.

Examples of the novel functions of the compound of formula (2) or saltsthereof include: suppression of T cell proliferation, suppression of Th2type cytokine production, and suppression of Th1 type cytokineproduction. Namely, the present invention relates to the use of thecompound of formula (2) or salts thereof as an agent that suppresses theaforementioned various allergic immune response induced by T cells thatplay a central role in allergic immune responses. Each of the functionis described in detail below.

The first novel function of the compound of formula (2) or salts thereofincludes the suppression of T cell proliferation. The in vitro optimalconcentration required to suppress T cell proliferation can bedetermined based either on or by referring to the concentration ofepinastine hydrochloride. The in vitro epinastine hydrochloride requiredto suppress T cell proliferation is 10 μM to 200 μM, preferably 20 μM to200 μM.

As the second novel function of the compound of formula (2) or saltsthereof, the ability to suppress the production of Th2 type cytokinescan be mentioned. The in vitro optimal concentration required for thesuppression of Th2 type cytokine production can be determined basedeither on or by referring to the concentration of epinastinehydrochloride. The in vitro epinastine hydrochloride concentrationrequired to suppress the production of Th2 type cytokines is 10 μM to200 μM, preferably 20 μM to 200 μM, and even more preferably 100 μM to200 μM.

The ability to suppress the production of Th1 type cytokines can beexemplified as the third novel function of the compound of formula (2)or salts thereof. Th1 type cytokines are produced from T helper celltype 1, and are involved in cellular immunity and the symptoms oforgan-specific autoimmune diseases (e.g., autoimmune thyropathy,multiple sclerosis, etc.) and inflammatory diseases (e.g., Crohn'sdisease, inflammatory bowel disease, rheumatoid arthritis, etc.).Psoriasis is also a Th1 type cytokine disease. Examples of Th1 typecytokines include IL-2 and IFN-γ. The identification and quantificationof these cytokines can be performed using ELISA wherein antibodiesagainst the cytokines are immobilized. The optimal in vitroconcentration required for the suppression of Th1 type cytokineproduction can be determined based either on or by referring to theconcentration of epinastine hydrochloride. The in vitro epinastinehydrochloride concentration required to suppress the production of Th1type cytokines is 10 μM to 200 μM, preferably 20 μM to 200 μM, and evenmore preferably 100 μM to 200 μM.

The optimal concentrations of the compound of formula (2) or saltsthereof required to suppress the various allergic immune responsesdescribed above used in vivo (in individuals such as animals) and thoseused for treating human diseases can be readily determined by oneskilled in the art using the aforementioned concentrations in an invitro system as standard or reference, and using the analytical methodspresented in the Examples.

As described above, the compound of formula (2) or salts thereof can beused to suppress allergic immune responses, such as T cellproliferation, Th2 type cytokine production, and Th1 type cytokineproduction, as well as inflammatory reactions arising from the allergicimmune response. Therefore, epinastine hydrochloride can be used as anagent to treat diseases involving these allergic immune responses.

Examples of these diseases include allergic rhinitis, bronchial asthma,atopic dermatitis, psoriasis vulgaris, autoimmune diseases involvingimbalance of Th1/Th2 cytokines (e.g., systemic lupus erythematosus,rheumatic arthritis, etc.), Crohn's disease, inflammatory bowel disease,and multiple sclerosis.

Allergic immune response suppressing agents comprising, as activeingredient, ebastine, carebastine, or the compound of formula (2) orsalts thereof may be used together with additives such as an excipient.Ebastine, carebastine, and the compound of formula (2) or salts thereofmay be administered either orally or parenterally. Examples of dosageforms include tablets, capsules, granules, powders, and injections. Whenadministered orally as tablets, capsules, granules, powders or such, thefollowing may be added as needed: excipients such as lactose,crystalline cellulose, starch, and vegetable oil; lubricants such asmagnesium stearate and talc; binders such as hydroxypropyl cellulose andpolyvinylpyrrolidone; disintegrators such as carboxymethylcellulosecalcium and low-substituted hydroxypropyl methylcellulose; coatings suchas hydroxypropyl methylcellulose, Macrogol, and silicone resin; andfilm-forming agents such as gelatin membranes. The formulation of theseagents can be performed according to conventional methods ordinary usedby those skilled in the art. In addition, ebastine, carebastine, and thecompound of formula (2) or salts thereof may also be incorporated into adrug delivery system or the like, if needed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of analyzing the effects of ebastine on T cellproliferation due to different stimulations. The reproducibility ofthese data was confirmed by five independent analyses using samplesderived from different donors.

FIG. 2 shows the results of analyzing the effects of carebastine (A) andepinastine hydrochloride (B) on T cell proliferation due to differentstimuli.

FIG. 3 shows the results of analyzing the effects of cetirizinehydrochloride (A) and ketotifen fumarate (B) on T cell proliferation dueto different stimuli.

FIG. 4 shows the effects of ebastine on the production of IL-2 (A) andIFN-γ (B) from peripheral blood T cells upon application of differentstimuli. The reproducibility of these data was confirmed by fiveindependent analyses using samples derived from different donors.

FIG. 5 shows the effects of carebastine on the production of IL-2 (A)and IFN-γ (B) from peripheral blood T cells upon application ofdifferent stimuli.

FIG. 6 shows the effects of epinastine hydrochloride on the productionof IL-2 (A) and IFN-γ (B) from peripheral blood T cells upon applicationof different stimuli.

FIG. 7 shows the effects of cetirizine hydrochloride on the productionof IL-2 (A) and IFN-γ (B) from peripheral blood T cells upon applicationof different stimuli.

FIG. 8 shows the effects of ketotifen fumarate on the production of IL-2(A) and IFN-γ (B) from peripheral blood T cells upon application ofdifferent stimuli.

FIG. 9 shows the effects of ebastine on the production of IL-4 (A) andIL-5 (B) from peripheral blood T cells upon application of differentstimuli. The reproducibility of these data was confirmed by fiveindependent analyses using samples derived from different donors.

FIG. 10 shows the effects of carebastine on the production of IL-4 (A)and IL-5 (B) from peripheral blood T cells upon application of differentstimuli.

FIG. 11 shows the effects of epinastine hydrochloride on the productionof IL-4 (A) and IL-5 (B) from peripheral blood T cells upon applicationof different stimuli.

FIG. 12 shows the effects of cetirizine hydrochloride on the productionof IL-4 (A) and IL-5 (B) from peripheral blood T cells upon applicationof different stimuli.

FIG. 13 shows the effects of ketotifen fumarate on the production ofIL-4 (A) and IL-5 (B) from peripheral blood T cells upon application ofdifferent stimuli.

FIG. 14 shows the effects of ebastine on the production of IL-6 (A) andTNF-α (B) from peripheral blood T cells upon application of differentstimuli. The reproducibility of these data was confirmed by fiveindependent analyses using samples derived from different donors.

FIG. 15 shows the effects of carebastine on the production of IL-6 (A)and TNF-α (B) from peripheral blood T cells upon application ofdifferent stimuli.

FIG. 16 shows the effects of epinastine hydrochloride on the productionof IL-6 (A) and TNF-α (B) from peripheral blood T cells upon applicationof different stimuli.

FIG. 17 shows the effects of cetirizine hydrochloride on the productionof IL-6 (A) and TNF-α (B) from peripheral blood T cells upon applicationof different stimuli.

FIG. 18 shows the effects of ketotifen fumarate on the production ofIL-6 (A) and TNF-α (B) from peripheral blood T cells upon application ofdifferent stimuli.

FIG. 19 shows the inhibitory effects of ebastine (A) and carebastine (B)on T cell migration through the vascular endothelial cells due to PHAactivation. The reproducibility of (A) was confirmed by threeindependent analyses using samples derived from different donors.

FIG. 20 shows the effects of epinastine hydrochloride (A), cetirizinehydrochloride (B), and ketotifen fumarate (C) on T cell migrationthrough the vascular endothelial cells due to PHA activation.

FIG. 21 shows photographs indicating the effects of ebastine on theadhesion of PHA-stimulated T cells to endothelial cells. Treatments wereperformed with 0 μM, 1.0 μM, and 5.0 μM ebastine, from the top to bottompanels, respectively.

FIG. 22 shows the effects of ebastine on the production of IL-6 (A) andTNF-α (B) from macrophages upon stimulation with LPS. Thereproducibility of these data was confirmed by five independent analysesusing samples derived from different donors.

FIG. 23 shows the effects of carebastine on the production of IL-6 (A)and TNF-α (B) from macrophages upon stimulation with LPS.

FIG. 24 shows the effect of epinastine hydrochloride on the productionof IL-6 (A) and TNF-α (B) from macrophages upon stimulation with LPS.

FIG. 25 shows the effects of cetirizine hydrochloride on the productionof IL-6 (A) and TNF-α (B) from macrophages upon stimulation with LPS.

FIG. 26 shows the effects of ketotifen fumarate on the production ofIL-6 (A) and TNF-α (B) from macrophages upon stimulation with LPS.

FIG. 27 shows photographs indicating the effects of ebastine on Cas-Land FAK tyrosine phosphorylations in PHA-stimulated T cells.

BEST MODE FOR CARRYING OUT THE INVENTION

Herein below, the present invention will be specifically described usingExamples; however, it is not to be construed as being limited thereto.

EXAMPLE 1 Effects of H1 Blockers on T Cell Proliferation Via DifferentActivation Pathways

All H1 blockers used for this comparative study belong to thelong-acting second-generation H1 blockers recently used in Japaneseclinical practice. Specifically, these included ebastine (trade nameEbastel), its active metabolite carebastine, epinastine hydrochloride(Alesion), and cetirizine hydrochloride (Zyrtec), all confirmed to beeffective as a single dose per day to improve compliance by patients. Asa control, ketotifen fumarate (Zaditen) which is said to have a highselectivity and affinity to H1 receptors among conventional H1 blockerswas added, and in total, five types of H1 blockers were examined.

The maximum blood concentration of ebastine is 0.03 μM, and that ofcarebastine, the active metabolite of ebastine, is 0.1 μM. Epinastinehydrochloride, cetirizine hydrochloride, and ketotifen fumarate act asthe same structure even after absorption via the intestinal tract, andtheir maximum blood concentrations are 0.1 μM, 0.5 μM, and 0.01 μM,respectively. The present experiments were performed using followingconcentrations: ebastine (0.1 μM to 10.0 μM), carebastine (1.0 μM to100.0 μM), epinastine hydrochloride (1.0 μM to 100.0 μM), cetirizinehydrochloride (5.0 μM to 500.0 μM), and ketotifen fumarate (0.1 μM to10.0 μM).

In order to analyze the effects of H1 blockers on T cell proliferation,three different costimulation pathways were used. The first pathway wasPMA, the second pathway stimulation on CD28 that is one of therepresentative T cell costimulation molecules, and the third pathwaystimulation on CD26 that is expressed on CD4⁺ memory T cells. Thesimulation on CD3 was applied together with all of these stimulations.The stimulation on CD3, CD26, and CD28 was performed using monoclonalantibodies OKT3, 1F7 (Morimoto C et al., 1989, supra), and 4B10 (immi CDet al., 1991, supra), respectively. OKT3 was purchased from the AmericanTissue Culture Collection (ATCC, Rockville, Md.), while 4B10 and 1F7were purified from mouse ascites of hybridomas established in thepresent inventors' laboratory (Gimmi C D et al., 1991, supra; Morimoto Cet al., 1989, supra).

Specifically, human peripheral blood mononuclear cells (PBMC) wereisolated from the peripheral blood of normal healthy persons by specificgravity centrifugation using Ficoll/Paque (Pharmacia Biotech Inc.,Piscataway, N.J.). Unfractionated mononuclear cells were separated usingthe E-rosette positive (E⁺) method and used as unstimulated T cells. Themononuclear cells were adhered to a plastic plate for 24 hours at 37°C., and then removed by culturing for one hour in 5 mM L-leucine methylester HCL (Sigma Chemical Co.) solution. A portion of the recoveredcells was stained with anti-CD3 antibodies and anti-CD14 antibodies, andthe purity of the cells was confirmed by flow cytometry. As a result,97% or more of the cells were found to be CD3 positive, while 1% or lessof the cells were CD14 positive. The cells obtained with theaforementioned purity were used as T cells.

Phosphate-buffer saline (PBS) (100 μl) containing either one of themonoclonal antibodies, OKT3 (0.05 μg/ml), 1F7 (1 μg/ml), or 4B10 (2μg/ml), was coated in triplicate according to a known method (Dang N Het al., 1990, supra) on flat-bottomed 96-well plates (Coster, Cambridge,Mass.), and the plates were then incubated overnight at 4° C. Beforeuse, these plates were washed once with PBS (200 μl). The highlypurified T cells (1×10⁵) described above were resuspended in RPMI medium(200 μl) containing 10% human AB serum and ebastine of four differentconcentrations (0 μM, 1 μM, 5 μM, and 10 μM). In order to evaluate PMAstimulation, a cell suspension containing PMA (5 ng/ml) was poured intothe wells coated with OKT3. The cells were cultured for three days underwet conditions at 37° C. and 5% CO₂. The cells were stimulated for eighthours with 1 μCi ³H-thymidine (ICN Radiochemicals, Irvine, Calif.) perwell, recovered on a glass fiber filter (Wallac, Turku, Finland) toquantify the incorporated radioactivity with a liquid scintillationcounter (Wallac).

As shown in FIGS. 1 to 3, the level of inducing T cell proliferationwith the stimulation on CD3 alone was low. Conspicuous T cellproliferation was observed when the stimulation on CD3 was combined withadditional second stimulation on CD26, CD28, or PMA. Under theseconditions, ebastine clearly inhibited T cell proliferation in aconcentration-dependent manner. Under each of the costimulationconditions and at all of the concentrations used in this Example,cetirizine hydrochloride and ketotifen fumarate hardly inhibited T cellproliferation (FIG. 3). On the other hand, ebastine (1 μM, 5 μM, and 10μM), carebastine (1 μM, 10 μM, and 100 μM), and epinastine hydrochloride(1 μM, 10 μM, and 100 μM) inhibited T cell proliferation by 10% to 50%(FIGS. 1 and 2) under each of the costimulation conditions. T cellproliferation induced by the anti-CD26 antibody tended to exhibit aslight resistance to the inhibition by ebastine. Moreover, at ebastineconcentrations of 20 μM to 50 μM, nearly 100% of T cell proliferationwas inhibited in a certain donor.

The inventors analyzed whether or not the above results were due to theside effect of cell toxicity of ebastine, carebastine, and epinastinehydrochloride by evaluating the cell survival rate through a trypan blueexclusion test. The T cell survival rate was 95% or higher at eachmeasurement point in all experiments. This indicates that the inhibitoryeffect of ebastine, carebastine, and epinastine hydrochloride was notdue to their side effect of cell toxicity.

EXAMPLE 2 Effects of H1 Blockers on Th1 Type Cytokine Production

In order to analyze whether or not H1 blockers have an inhibitory effecton cytokine production induced by the various costimulations describedabove, the present inventors measured Th1 type cytokines (IL-2, andIFN-γ) in the culture supernatant induced by the various costimulationsdescribed above.

The antibody-coated plates and T cells were prepared similarly as inExample 1 except that the OKT3 concentration was 0.5 μg/ml. The cytokineproduction by T cells was analyzed in triplicate using 96-wellflat-bottomed plates by the same method employed in the T cellproliferation analysis described above. After culturing for 24 hours,the culture supernatant was tested using ELISA kit (IL-2: BiosourceInternational, Camarillo, Calif.; IFN-γ: R&D systems, Minneapolis,Minn.) to quantify IL-2 and IFN-γ.

As shown in FIGS. 4A and 5A, ebastine and carebastine did not inhibitIL-2 production under any of the costimulation conditions even at themaximum concentrations (10 μM for ebastine, and 100 μM for carebastine).In addition to IL-2, ebastine and carebastine did not have a cleareffect on IFN-γ production at any of the concentrations of 0.1 μM to 10μM and 1 μM to 100 μM, respectively (FIGS. 4B and 5B). On the otherhand, epinastine hydrochloride suppressed both IL-2 and IFN-γ productiondue to each costimulation in a dose-dependent manner (FIG. 6). Inaddition, cetirizine hydrochloride and ketotifen fumarate did not have aclear effect on IL-2 and IFN-γ production due to each costimulation(FIGS. 7 and 8). Therefore, these results indicated that ebastine andcarebastine do not inhibit Th1 type cytokine production under anycostimulation conditions.

EXAMPLE 3 Effects of H1 Blockers on Th2 Type Cytokine Production

Th2 type CD4 T cells play an important role in allergic diseases.Therefore, the present inventors analyzed the effects of ebastine on Th2type cytokines (IL-4, and IL-5) induced by the various costimulationsdescribed above. This analysis was performed similarly as in Example 2except that ELISA (IL-4: Biosource International, Camarillo, Calif.;IL-5: R&D systems, Minneapolis, Minn.) was used to quantify IL-4 andIL-5.

As shown in FIGS. 9A, 10A, and 11A, ebastine, carebastine, andepinastine hydrochloride inhibited IL-4 production under eachcostimulation condition in a concentration-dependent manner. Inparticular, ebastine nearly completely inhibited IL-4 production from Tcells at 10 μM. Furthermore, as shown in FIGS. 9B, 10B, and 11B,ebastine, carebastine, and epinastine hydrochloride inhibited IL-5production in a concentration-dependent manner under each of thecostimulation conditions. In comparison to the effect of ebastine onIL-4 production from T cells, IL-5 production was inhibited by 50% ormore at an ebastine concentration of 5 μM, indicating IL-5 productionbeing more sensitive to ebastine. This result was reproduced in fiveindependent donors. Moreover, at a concentration of 20 μM, IL-4 and IL-5productions were 100% suppressed in all donors. In addition, in thesuppression of cytokine production, the carebastine dose was higher thanthat of ebastine. On the other hand, both cetirizine hydrochloride andketotifen fumarate did not have a clear effect on IL-4 and IL-5productions due to each costimulation (FIGS. 12 and 13).

EXAMPLE 4 Effects of H1 Blockers on Inflammatory Cytokine Production

The present inventors analyzed the effects of H1 blockers on theproduction of inflammatory cytokines, such as IL-6 and TNF-α, from Tcells due to the various costimulation pathways described above. Thisanalysis was performed similarly as in Example 2 above except for theuse of an ELISA kit for measuring IL-6 and TNF-α (R&D systems,Minneapolis, Minn.).

As shown in FIGS. 14 and 15, ebastine and carebastine inhibited, in aconcentration-dependent manner, the inflammatory cytokines IL-6 andTNF-α from T cells under each of the costimulation conditions. Ebastineachieved 100% suppression at concentrations from 10 μM to 20 μM. Thisresult was reproduced in three independent donors. On the other hand,regarding epinastine hydrochloride, no clear effect of suppressing IL-6and TNF-α production was found by any of the costimulations (FIG. 16).In addition, cetirizine hydrochloride and ketotifen fumarate did nothave a clear effect on IL-6 and TNF-α productions due to eachcostimulation (FIGS. 17 and 18).

EXAMPLE 5 Effects of H1 Blockers on Transendothelial T Cell Migration

In order to determine the effect of ebastine on T cell permeation, thepresent inventors analyzed vascular transendothelial migration ofPHA-stimulated T cells in vitro. Since fresh resting-stage T cellshardly migrate by permeating a monolayer of endothelial cells, these Tcells were activated using PHA (10 μg/ml) prior to performingtransendothelial migration analysis.

Specifically, the analysis of transendothelial migration was performedby slightly modifying a known method (Iwata S, Yamaguchi N, Munakata Y,Ikushima H, Lee J F, Hosono O, Schlossman S F, Morimoto C. “CD26/DPPIVdifferentially regulates the chemotaxis of T cells and monocytes towardRANTES. Possible mechanism for the switch from innate to acquired immuneresponse.” Int Immunol. 11:417-426, 1999). Human endothelial cell strainECV304 was obtained from the American Tissue Culture Collection (ATCC;Rockville, Md.) and cultured for 48 hours on Transwell cell cultureinserts (Corning Costar, Cambridge, Mass.) with a pore size of 3.0 μm.The cells were loaded onto a 24-well plastic plate for cell culture(FALCON), and mixed with H1 blockers at various concentrations usingTranswell assay medium (RPMI1640 medium, BSA 0.6%, PenicillinG/Streptomycin 1%, HEPES 50 mM). Two-hundred μl of PHA-stimulated Tcells (1×10⁷/ml) were then placed in the upper chamber and 600 μl ofassay medium was placed in the lower chamber, and migration wasperformed for eight hours at 37° C. under 5% CO₂. After recovery of thecells, the number of cells was counted for one minute by flow cytometry(FACSCalibur, Nippon Becton-Dickinson, Tokyo, Japan).

As shown in FIG. 19A, T cell migration was markedly inhibited in thepresence of ebastine in a concentration-dependent manner. It isnoteworthy that the inhibition of T cell migration reached a plateaulevel at a concentration of 5 μM. In addition, T cell migration was alsosuppressed in the presence of carebastine in a concentration-dependentmanner (FIG. 19B). Conversely, when ECV cells were pretreated withebastine of various concentrations for 48 hours, washed and then exposedto PHA-stimulated T cells, ebastine did not show effect on the T cellmigration via ECV cells even at the maximum concentration used in theexperiment (10 μM) (not shown). Thus, it was shown that T cell migrationis suppressed when ebastine is present at the time of T cell migrationanalysis. On the other hand, epinastine hydrochloride, cetirizinehydrochloride, and ketotifen fumarate did not have a clear effect on themigration of PHA-stimulated T cells (FIG. 20).

Ebastine suppressed PHA-stimulated T cell migration in a dose-dependentmanner. Thus, the adhesion of PHA-stimulated T cells to endothelialcells was analyzed in order to elucidate the action mechanism of thesuppression of T cell migration by ebastine. PHA-stimulate T cells werereacted for 30 minutes with the fluorescent dye3′-O-Acetyl-2′,7′-bis(carboxyethyl)-4- or 5-carboxyfluorescein,diacetoxymethyl ester (BECEF-AM), treated with H1 blocker, ebastine (1.0μM to 5.0 μM), and then plated on a culture dish to which endothelialcells were adhered. After six hours, the culture dish was washed threetimes with PBS. Because of observation under a fluorescent microscope,ebastine was found to suppress the adhesion of PHA-stimulated T cells toendothelial cells in a dose-dependent manner.

Moreover, cell surface staining experiments were performed usingmonoclonal antibodies against various adhesion molecules on T cells,such as CD11a, CD29, CD44, and CD47, activation antigens such as CD25and CD26, and ligands of the adhesion molecules on endothelial cellmembranes such as ICAM-1 and VCAM-1.

PHA-stimulated T cells and endothelial cells were cultured for 6 hoursin the presence of ebastine (1.0 μM to 5.0 μM) at 37° C. under 5% CO₂,and antibodies to various cell surface molecules (CD3, CD11a, CD25,CD26, CD29, CD44, CD47, ICAM-1, and VCAM-1) were reacted with the cellsfor 30 minutes at 4° C. The cells were washed with PBS containing 2% FBSand 0.02% sodium azide (Sigma), and further reacted with FITC-labeledgoat anti-mouse IgG antibody (Sigma) for 30 minutes at 4° C. Washedthree more times with PBS, and then the cell fluorescence intensity wasmeasured by flow cytometry. The antibodies used to identify T cellsurface molecules were: anti-CD3 antibody (OKT3), anti-CD11a antibody(BD PharMingen, NJ, USA), anti-CD25 antibody (BD PharMingen), anti-CD26antibody (1F7), anti-CD29 antibody (4B4), anti-CD44 antibody (BDPharMingen), and anti-CD47 antibody (BD PharMingen); and those for theligands of the adhesion molecules on the endothelial cell surfaces were:anti-ICAM-1 antibody and anti-VCAM-1 antibody. The results are presentedin Tables 1 and 2. TABLE 1 Ebastine concentration 0 μM 1 μM 5 μM (-) 0.30.3 0.3 CD3 10.6 ± 0.26 10.6 ± 0.2: N.S. 10.9 ± 0.26: N.S. CD11a 53.1 ±0.44 53.8 ± 0.44: N.S. 53.9 ± 0.4: N.S. CD25 18.2 ± 0.56 18.3 ± 0.35:N.S. 16.5 ± 0.26: ** CD26 102.0 ± 0.69  99.1 ± 0.36: ** 87.8 ± 0.36: ***CD29 28.0 ± 0.56 25.0 ± 0.26: * 22.1 ± 0.36: *** CD44 48.7 ± 0.62 47.6 ±0.30: N.S. 47.9 ± 0.36: N.S. CD47 14.5 ± 0.35 11.4 ± 0.17: ** 10.0 ±0.26: ****: P < 0.05,**: P < 0.01,***: P < 0.001,N.S.: Not Significant

TABLE 2 Ebastine concentration 0 μM 1 μM 5 μM (-) 0.3 0.3 0.3 ICAM-120.3 ± 0.41 20.7 ± 0.52 20.4 ± 0.26 VCAM-1 0.3 0.3 0.3

After treatment with ebastine for eight hours, among all the adhesionmolecules and activation antigens tested on the T cells, the expressionof CD26, CD29, and CD47 was markedly effected (Table 1). At the ebastineconcentration of 5 μM, the expression levels of CD26, CD29, and CD47decreased by approximately 14%, 21%, and 31%, respectively. On the otherhand, the expression levels of CD3, CD11a, CD25, and CD44 werecompletely unaffected (Table 1). In addition, ebastine had no effect onthe fluorescence intensity of ligands, ICAM-1 and VCAM-1, of theadhesion molecules on the endothelial cell membrane (Table 2). SinceCD26, CD29, and CD47 have been reported to be involved in cellmigration, these results partially explain the inhibitory effect ofebastine on T cell migration.

EXAMPLE 6 Effects of H1 Blockers on Inflammatory Cytokine Production byMacrophages

Macrophages participate in the early stages of the host-protectionmechanism against infection and in the local regulation ofimmune/inflammatory reactions (Johnston R B Jr. “Monocytes andmacrophages.” N Eng J Med. 318:747-752, 1988). Therefore, the effects ofebastine on the production of inflammatory cytokines such as IL-6 andTNF-α were analyzed.

Specifically, in order to produce IL-6 and THF-α from macrophages,peripheral blood mononuclear cells were adhered to a plastic plate toisolate and recover macrophages. These macrophages (1×10⁶/ml) weresuspended in RPMI containing 10% FCS and stimulated with LPS (1 μg/ml).After culturing for eight hours at 37° C. under 5% CO₂, the culturesupernatant was recovered and the amount of produced IL-6 and TNF-αquantified using an ELISA kit for measuring IL-6 and TNF-α (R&D systems,Minneapolis, Minn.).

As shown in FIGS. 22 and 23, ebastine and carebastine suppressed, in adose-dependent manner, the production of IL-6 and TNF-α from macrophagesdue to LPS stimulation. In particular, at an ebastine concentration of 1μM, the production of the above cytokines was suppressed by 50% or more,and at 10 μM, the production was nearly completely inhibited (FIG. 22).Thus, ebastine was also demonstrated to inhibit the production ofinflammatory cytokines such as IL-6 and TNF-α from macrophages. On theother hand, no inhibition on the production of IL-6 and TNF-α frommacrophages due to LPS stimulation was observed with epinastinehydrochloride, cetirizine hydrochloride, and ketotifen fumarate (FIGS.24 to 26).

EXAMPLE 7 Suppression of Cas-L Tyrosine Phosphorylation inPHA-Stimulated T Cells by Ebastine

Ebastine suppressed T cell functions following T cell proliferation,such as cytokine production, cell migration, and adhesion, as well asthe expression of β1 integrin, CD29. Thus, the present inventorsexamined the effects of ebastine on the tyrosine phosphorylation of CD29integrin-related signal transduction molecules. PHA-stimulated T cellstreated with ebastine (0.1 μM to 1.0 μM) were washed three times withIscove's modified Dulbecco's medium (IMDM) and then solubilized bystanding still for 30 minutes at 4° C. in lysis buffer (1% NP40, 140 mMNaCl, 50 mM Tris, 5 mM EDTA, 5 mM NaF, 1 μg/ml aprotinin, 1 μg/mlleupeptin, 1 μg/ml pepstatin A, 1 mM PMSF, 1 mM sodium vanadate, pH7.5). The cell lysate was centrifuged for 30 minutes at 15,000 rpm and4° C. to recover a cell extract from the supernatant. Tyrosinephosphorylation was analyzed by Western blotting of the cell extractusing anti-phosphorylated tyrosine antibody. Among the bands obtained bythe analysis of the cell extract, Cas-L, a signal transduction moleculedownstream of β1 integrin, and FAK, involved in Cas-L phosphorylation,were analyzed by immunoprecipitation and Western blotting. Specifically,anti-Cas-L antibody or anti-FAK antibody was added to the above cellextract and reacted overnight at 4° C. Protein A beads were then addedand allowed to react for four hours at 4° C. The reaction solution wascentrifuged for five minutes at 3,000 rpm and 4° C., and the beads werewashed with wash buffer (0.5% NP40, 140 mM NaCl, 50 mM Tris, 5 mM EDTA,5 mM NaF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1mM PMSF, 1 mM sodium vanadate, pH 7.5). This operation was repeatedthree times, sample buffer (1% SDS) was added to the beads from whichthe wash buffer was removed, and was reacted for five minutes at 95° C.The reaction solution was centrifuged and the supernatant was used assamples. An 8% acrylamide gel was prepared, and the samples weresubjected to electrophoresis in an electrophoresis buffer (10% SDS, 250mM Tris, 1.92 M glycine) for 120 minutes at 100V. After theelectrophoresis, the bands were transferred using a transfer buffer (250mM Tris, 1.92 M glycine, 20% methanol) on a PVDF membrane (Millipore)for 80 minutes at 100V and 4° C. After the transfer, the membrane wassubjected to blocking in TBS-T (0.05% Tween20, 50 mM Tris, 150 mM NaCl,pH 7.5) supplemented with 5% milk for 60 minutes, and then primaryantibody (1 μg/ml) was reacted in TBS-T for 60 minutes. After washingthe membrane with TBS-T, HRP-labeled goat anti-mouse IgG antibody(1:10,000) was reacted as the secondary antibody for 60 minutes in TBS-Tsupplemented with 0.5% milk. The membrane was washed with TBS-T, reactedin ECL solution, and then exposed to a film for autoradiography.

As a result, ebastine was found to slightly suppress the tyrosinephosphorylation of FAK in PHA-stimulated T cells, and to suppress thetyrosine phosphorylation of Cas-L in a dose dependent manner (FIG. 27).Thus, as one mechanism of the suppression of cell migration, ebastinewas thought to suppress cell migration through the suppression oftyrosine phosphorylation of Cas-L.

As described above, ebastine inhibited the production of Th2 cytokines,IL-4 and IL-5, and the inflammatory cytokines IL-6 and TNF-α from Tcells. This inhibition was specific, and did not influence theproduction of the Th1 cytokines, IL-2 and IFN-γ. In addition, ebastinealso suppressed T cell migration, and further the production ofinflammatory cytokines IL-6 and TNF-α from macrophages.

Ebastine is a novel H1 blocker that does not induce drowsiness, andutilizing this effect, has been clinically used for allergic rhinitis,urticaria, and the like (Ohtani H, Sato H, Iga T, Kotaki H, Sawada Y.“Pharmacokinetic-pharmacodynamic analysis of the arrhythmogenic potencyof a novel antiallergic agent, ebastine, in rats.” Biopharm DrugDispose. 20:101-106, 1999; Matsuda M, Sakashita M, Mizuki Y, YamaguchiT, Fujii T, Sekine Y. “Comparative pharmacokinetics of the histamineH1-receptor antagonist ebastine and its active metabolite carebastine inrats, guinea pigs, dogs and monkeys.” Arzheim Forsch Drug Res. 44:55-59,1994; Storms W W, 1966, supra; Kalis B, 1996, supra). Interactionsbetween histamines and histamine H1 receptors induce pruritus and paindue to a series of physiological effects like contraction of importantbronchial smooth muscles and increased vascular permeable stimulation ofperipheral nerve endings, and also reflexively induce bronchial stenosisand coughing from stimulation of vagus nerve endings (Rimmer S J, ChurchM K. “The pharmacology and mechanisms of action of histamineH1-antagonists.” Clin Exp Allergy. 20 Suppl 2:3-17, 1990).

The effects of ebastine have been reported to be mainly due to theinhibition of the release of histamines from basophils and mast cells(Rimmer S J, et al., 1990, supra; Birendia E. “Ebastine in contextintroduction.” Drugs 52 Suppl 1:1-7, 1996). Moreover, ebastine has beenreported to exhibit blocking of the release of anti-IgE-inducedprostaglandin D2 and leukotriene C4/D4 from human nasal polyp cellsafter in vitro antigen administration (Campbell A, Michel F-B,Bremard-Qury C, Crampette L, Bousquet J. “Overview of allergicmechanisms. Ebastine has more than an antihistamine effect.” Drugs 52(Suppl 1):15-19, 1996). Furthermore, ebastine has been suggested topossess effects in addition to the blocking of histamine H1 receptors.Herein, effective novel effects of ebastatine are disclosed.

In hypersensitive responses, helper CD4 T cells play an important rolein inducing allergic inflammatory responses. That is, cytokineproduction by Th2 type cells is essential to develop and maintainhypersensitive inflammatory responses (Kapsenberg M, et al., 1991,supra; Romagnani S., 1994, supra). The transportation of activated Tcells to inflammatory sites is strictly controlled by a large number ofmolecules, particularly adhesive molecules and chemokines (Butcher E Cand Picker L J. “Lymphocyte homing and homeostasis.” Science. 272:60-66,1996). The selective recruiting of subsets of CD4⁺ effector T cells toinflammatory sites is thought to contribute to the development ofdifferent pathological symptoms. Activated Th2 type CD4 T cells thatproduce IL-4, IL-5, and IL-13 are detected in allergic inflammatorysites, bronchoalveolar lavage, and brachial biopsies of atopic andnonatopic asthmatics. Such cells have recently been positioned at thecenter of the action mechanism in the pathophysiology of asthma(Robinson D S et al. “Predominant Th2-type bronchoalveolar lavageT-lymphocyte population in atopic asthma.” N Engl J Med. 326:298-304,1992; Ying S et al. “Expression of IL-4 and IL-5 mRNA and proteinproducts by CD4⁺ and CD8⁺ T cells, eosinophils and mast cells inbronchial biopsies obtained from atopic and nonatopic (intrinsic)asthmatics.” J Immunol. 158:3539-3544, 1997). Therefore, Th2 type CD4 Tcells and cytokines thereof are regarded as important targets fortreatment. The Examples demonstrate that ebastine can inhibit, in aconcentration-dependent manner, the production of Th2 type cytokinessuch as IL-4 and IL-5 due to various costimulation conditions.

On the other hand, ebastine does not affect the production of Th1 typecytokines such as IL-2 and IFN-γ. In addition, terfenadine which is ahistamine H1 receptor antagonist having a chemical structure similar tothat of ebastine has also been reported to have a similar effect(Munakata Y, Umezawa Y, Iwata S, Dong R-P, Yoshida S, Ishii T, MorimotoC. “Specific inhibition of Th2-type cytokine production from humanperipheral T cells by terfenadine in vitro.” Clin Exp Allergy.29:1281-1286, 1999). However, since terfenadine induces occasionally QTinterval elongation and infrequently fatal acute ventricular arrhythmia(Woolsey R L. “Cardiac actions of antihistamines.” Ann Rev PharmacolToxicol. 36:233-252, 1996), it is currently not used in Europe, America,and Japan. On the other hand, ebastine does not have such severecardiovascular side effects and has been reported not to affect the QTinterval even if its dose is increased to five times from that used forthe treatment (Ohtani H, et al., 1999, supra; Gillen M S, Miller B,Chaikin P, Morganroth J. “Effects of supratherapeutic doses of ebastineand terfenadine on the QT interval.” Br J Clin Pharm. 52:201-204, 2001,Roberts D J. “Assessing the cardiac safety of ebastine.” Epilogue DrugSaf. 21 (Suppl I):89-92, 1999).

Terfenadine and other H1 receptor antagonists have been reported toinhibit eosinophil and neutrophil chemotaxis and reduce the expressionof ICAM molecules on epithelial cell lines (Negro-Alvarez J M, Funes E,Garcia Canovas A, Herhamdez J, Garcia-Selles F J, Pagan J A,Lopez-Sanchez J D. “Antiallergic properties of antihistamines.” AllergolImmunopathol. 24:177-183, 1996). However, it has not been revealedwhether these H1 antagonists inhibit T cell migration or not. On theother hand, as indicated by the above Examples, ebastine has been shownto inhibit T cell migration in a concentration-dependent manner. Thissuppression of T cell migration was due to the suppression of T celladhesion to endothelial cells via suppressing the expression ofadhesion-related T cell surface molecules CD26, CD29, and CD47.Furthermore, the research results of the present inventors alsosuggested suppression of tyrosine phosphorylation of Cas-L, which is adownstream signal of β1 integrin, as one of the mechanisms of thesuppression of T cell migration. β1 integrin transmits a diversity ofsignals via the activation of various target molecules due to tyrosinephosphorylation, thereby exhibiting its biological functions. Thetyrosine phosphorylation of the Cas-L positioned downstream of β1integrin has been suggested to play an important role in the T cellactivation and subsequent proliferation, cytokine production and celladhesion, and the onset of cell migration (Ohashi Y. J Cell Biol, 1995,supra; Kamiguchi K, J Immunol, 1999, supra). Therefore, the suppressionof Cas-L tyrosine phosphorylation by ebastine is strongly suggested asone of the suppression mechanisms of T cell migration.

Ebastine also inhibited the production of inflammatory cytokines such asIL-6 and TNF-α from human macrophages and T cells. Terfenadine has beenreported to inhibit the release of IL-4 and IL-13 from human basophils(Gibbs B F, Vollrath I B, Albrecht C, Amon U, Wolff H H. “Inhibition ofinterleukin-4 and interleukin-13 release from immunologically activatedhuman basophils due to the actions of anti-allergic drugs.”Naunyn-Schmiedebergs Arch Pharmacol. 357:573-578, 1998), and ebastinehas been reported to inhibit TNF-α, IL-8, and GM-CSF production fromnasal polyp cells (Campbell A, et al. 1996, supra). However, thesuppression of inflammatory cytokine production from macrophages and Tcells as described above has not been reported.

Macrophages are dominant cells in human airways, and they are one of themost plentiful cells in the lung parenchyma of both healthy persons andasthmatics (Johnston R B Jr., 1988, supra), and provide the largestnumber of functional factors (Johnston R B Jr. “Current concepts:immunology. Monocytes and macrophages.” N Engl J Med. 318:747, 1988).Macrophages synthesize and release a wide range of inflammatory factorssuch as oxygen, cytokines, and chemokines (Nathan C F “Secretaryproducts of macrophages.” J Clin Invest 79:319, 1987). The mainchemokines produced from lung macrophages are IL-6 and TNF-α (Marone G,Gentile M, Petraroli A, De Rosa N, Triggiani M. “Histamine-inducedactivation of human lung macrophages.” Int Arch Allergy Immunol.124:249, 2001; Amrani Y, Chen H, Panettieri R A Jr. “Activation of tumornecrosis factor receptor 1 in airway smooth muscle: a potential pathwaythat modulates bronchial hyper-responsiveness in asthma.” Respir Res.1:49, 2000).

According to a recent study, since tumor necrosis factor (TNF)-α isproduced in large amounts in the airways of asthmatics, it is suggestedto be involved in the onset of bronchial hypersensitive responses bydirectly changing the state of airway smooth muscle (ASM) (Amrani. Y,Chen H, Panettieri R A Jr. “Activation of tumor necrosis factor receptor1 in airway smooth muscle: a potential pathway that modulates bronchialhyper-responsiveness in asthma.” Respir Res. 1:49, 2000).

In addition, IL-6 is a cytokine with many functions that is released ininflammatory diseases. This cytokine is involved in the differentiationand maturation of human mast cells, and in the control of IgE synthesis(Hirano T. “Interleukin 6 and its receptor: ten years later.” Int RevImmunol. 16:249, 1998). Elevated IL-6 levels have been detected inblood, bronchoalveolar lavage fluid after bronchial treatment ofasthmatics, and in bronchial biopsies, which suggests elevated IL-6expression (Virchow J C Jr., Walker C, Hafner D, Kortsik C, Werner P,Matthys H, et al. “T cells and cytokines in bronchoalveolar lavage fluidafter segmental allergen provocation in atopic asthma.” Am J Respir CritCare Med. 151:960, 1995).

Cytokine networks and abnormalities thereof in autoimmune diseases areattracting attention in recent years. One factor of the onset ofautoimmune diseases is thought to be the Th1/Th2 imbalance induced bythe invasion of autoantigens or foreign antigens. Autoimmune diseasescan be broadly classified into Th1-dependent types, Th2-dependent types,and those in which both are involved (Ohta A, Nishimura T. “Cytokinenetworks and abnormalities thereof in autoimmune diseases.” The MedicalFrontline (in Japanese) 53(6):1338, 1998). As representative diseases,SLE is Th2 cytokine-dominant while chronic rheumatoid arthritis involvesboth Th1 and Th2 cytokines (Ohta A, The Medical Frontline, 1998, supra).

Based on the various findings and the Examples given above, suppressionof IL-6 and TNF-α production from T cells and macrophages by ebastine issuggested to be an important target for asthma treatment.

Furthermore, the antiallergic action of epinastine hydrochloride isthought to involve the suppression of the production of Th1 and Th2 typecytokines from T cells in addition to histamine antagonism.

In addition to its antihistamine effects, ebastine has been shown tohave novel functional effects on T cells and macrophages. Ebastineinhibited Th2 type cytokine production from T cells, T cell migration,and production of inflammatory cytokines (IL-6, TNF-α, etc.) from Tcells and macrophages. Therefore, ebastine may serve as an essentialagent for the treatment of immunoallergic diseases caused by T cells,including asthma, atopic dermatitis, Th2 type autoimmune diseases,systemic lupus erythematosus, myasthenia gravis, chronic GVHD,inflammatory diseases such as Crohn's disease, and so on. Moreover,chronic rheumatoid arthritis is triggered by the production ofinflammatory cytokines such as TNF-α and IL-6 from T cells andmacrophages at inflammatory sites, and T cell migration to inflammatorysites. Therefore, ebastine may be applicable for the treatment of thesediseases. In particular, recent reports showed that anti-TNF-αtreatments are effective on rheumatic arthritis and Crohn's disease(Taylor P C, Williams R O, Maini R N. “Immunotherapy for rheumatoidarthritis.” Curr Opin Immunol. 13:611, 2001; Kam L Y, Targan S R.“TNF-alpha antagonists for the treatment of Crohn's disease.” ExpertOpin Pharmacother. 1:615, 2000) and that IL-6 receptor antibodytreatments are also effective in the treatment of rheumatoid arthritis(Choy E H, Isenberg D A, Garrood T, Farrow S, Ioannou Y, Bird H, et al.“Therapeutic benefit of blocking interleukin-6 activity with ananti-interleukin-6 receptor monoclonal antibody in rheumatoid arthritis:A randomized, double-blind, placebo-controlled, dose-escalation trial.”Arthritis Rheum. 46(12):3143, 2002). Thus, ebastine may be used in thetreatment of rheumatoid arthritis.

On the other hand, epinastine hydrochloride is reported to have anefficacy of 47.0% in the improvement of bronchial asthma to a mediumdegree or greater, and its administration for psoriasis vulgarisaccompanying pruritus has been clinically approved (according to theInterview Form for epinastine hydrochloride). Regarding the clinicaleffects of epinastine hydrochloride, in addition to the antagonisticeffect to H1 receptors, it is reported to influence the cytokineproduction of peripheral blood eosinophils and mononuclear cells (SagaraH. “Effects of epinastine on eosinophils.” Allergology (in Japanese).12(6):587, 2001; Kojima Y, Yoshikawa Y, Nishibe A, Kaneko F. “Effects ofantiallergic agents on cytokine production by peripheral bloodmononuclear cells in atopic dermatitis patients.” Jpn J Dermatology.106(4):395, 1996). According to the present invention, epinastinehydrochloride was shown to suppress T cell proliferation and Th1 and Th2cytokine production from T cells. This is thought to be one mechanismexplaining its efficacy, since psoriasis vulgaris in particular isassumed to be a Th1 type disease and epinastine hydrochloride iscurrently used against it.

However, H1 blockers cetirizine hydrochloride and ketotifen fumaratewere not found to affect the functions of T cells and macrophages.

According to a comparison of the IC₅₀ values of inhibiting the ligandbinding of H1 blockers to H1 receptors using Chinese Hamster Ovary cellsthat express human recombinant H1 receptors, the value of ketotifenfumarate was 0.52±0.06 nM, that of epinastine hydrochloride was 1.1±0.16nM, and that of cetirizine hydrochloride was 11±0.67 nM (Nonaka H etal., 1998, supra). In addition, in a comparison of the IC₅₀ values ofthe effects on histamine-induced contraction in trachea samples excisedfrom guinea pigs, the value of ketotifen fumarate was 0.0025 μM, that ofcarebastine was 0.12 μM, and that of ebastine was >10 μM (according tothe Interview Form for ebastine). As described above, among the H1blockers used herein, ketotifen fumarate had the strongest affinity andantagonism to an H1 receptor. However, ketotifen fumarate showed noeffect on T cells and macrophages. Thus, H1 blockers were suggested toexert effects on immune cells through other than antihistaminic effectson histamine receptors.

In addition to H1 receptors, H1 blockers also bind to H2 receptorsalthough to a lesser extent. The IC₅₀ values (nM) in the inhibition ofligand binding to H2 receptors was 640±62, 2200±450, and >10,000 forepinastine hydrochloride, ketotifen fumarate, and cetirizinehydrochloride respectively (Nonaka H et al., 1998, supra). The abovereport was based on non-clinical experiments performed to supplement thefact that the binding specificity of cetirizine hydrochloride to H1receptors as an H1 blocker is higher than that of epinastinehydrochloride and ketotifen fumarate. In this report, no controlexperiments to ebastine were performed. Binding to H2 receptors iscurrently considered as only a side effect of H1 blockers, and thus,details regarding the binding of ebastine to H2 receptors have not beenreported. However, the comparison of the IC₅₀ values of the effects onhistamine-induced contraction in trachea samples excised from guineapigs and such showed that the affinity of ebastine (or carebastine) toH1 receptors is much less than that of ketotifen fumarate. Therefore,the specificity of ebastine (or carebastine) to H1 receptors may besmaller than ketotifen fumarate.

Histamine receptors in human T cells are mainly H2 receptors. Thepresent inventors previously reported that the H1 blocker terfenadinesuppresses cytokine production by T cells (Munakata et al., 1999). Inthe binding experiment to H2 receptors using terfenadine, the inhibitionrate to [³H]-tiotidine was 29% at maximum concentration (Uchida M, OmiN, Koga Y, Morooka S. “Pharmacological effects of cetirizine.” TheClinical Report (in Japanese). 28(7):1795-1812, 1994). According to thepresent invention, suppression of T cell cytokine production wasobserved for the H1 blockers ebastine and epinastine hydrochloride,which have a relatively low specificity to H1 receptors. This may be dueto the fact that they acted on H2 receptors on human T cells. In mice,histamine has been reported to enhance Th1 cytokine production via H1receptors, and conversely suppress both Th1 and Th2 cytokine productionvia H2 receptors (Jutel M, et al., Nature, 2001, supra). According tothe present invention, the effect of suppressing cytokine production viaH2 receptor stimulation demonstrated in mouse T cell systems wassuggested to also occur in human T cells.

INDUSTRIAL APPLICABILITY

As described above, ebastine and carebastine have been shown to inhibitT cell proliferation, Th2 type cytokine production, inflammatorycytokine production, and T cell migration under costimulationconditions, all in a concentration-dependent manner. Due to theseeffects, ebastine and carebastine are expected to serve as essentialagents for the treatment of immunoallergic diseases caused by T cells.Examples of diseases include asthma, atopic dermatitis, Th2 typeautoimmune diseases, and inflammatory diseases such as systemic lupuserythematosus, myasthenia gravis, chronic GVHD, Crohn's disease, andchronic rheumatoid arthritis.

In addition, epinastine hydrochloride has been shown to inhibit T cellproliferation, Th2 type cytokine production, and Th1 type cytokineproduction, all in a concentration-dependent manner. Due to theseeffects, epinastine hydrochloride is expected to serve as an essentialagent for the treatment of diseases such as allergy, asthma, psoriasisvulgaris, organ-specific autoimmune diseases involving Th1/Th2 cytokineimbalances (autoimmune thyropathy, and multiple sclerosis), inflammatorydiseases (Crohn's disease, inflammatory bowel disease, and rheumatoidarthritis), systemic autoimmune diseases (systemic lupus erythematosus,and myasthenia gravis), and chronic GVHD.

1. A compound having the formula (1):

(wherein, R is CH₃ or COOH), that is able to suppress an allergic immuneresponse involving an immunocyte, wherein said allergic immune responseis selected from the group consisting of: (A) proliferation of T cell;(B) production of Th2 type cytokine; (C) production of inflammatorycytokine; and (D) T cell migration.
 2. A compound having the formula (2)or salt thereof:

, that is able to suppress an allergic immune response involving animmunocyte, wherein said allergic immune response is selected from thegroup consisting of: (A) proliferation of T cell; (B) production of Th2type cytokine; and (C) production of Th1 cytokine.
 3. The compoundaccording to claim 1, wherein the inflammatory cytokine is produced froma T cell or macrophage.
 4. A composition comprising the compoundaccording to claim 1 or
 3. 5. A composition comprising the compound orsalts thereof according to claim
 2. 6. An agent for suppressing anallergic immune response involving an immunocyte that comprises, as anactive ingredient, the compound having the formula (1):

wherein said allergic immune response is selected from the group of: (A)proliferation of T cell; (B) production of Th2 type cytokine; (C)production of inflammatory cytokine; and (D) T cell migration.
 7. Anagent for suppressing an allergic immune response involving animmunocyte that comprises, as an active ingredient, the compound havingthe formula (2) or salts thereof:

wherein said allergic immune response is selected from the group of: (A)proliferation of T cell; (B) production of Th2 type cytokine; and (C)production of Th1 type cytokine.